[생물공정실험] 5주차 Spore sterilization for food processing 결과보고서

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1. Spore sterilization for food processing

이번 실험에서는 moist heat method 중 하나인 Pasteurization(저온살균)과 filteration method 중 하나인 membrane filter를 이용해 Clostridioides difficile 균을 sterilization해 보았다. C.difficile 균은 포자를 형성할 수 있는 혐기성 그람양성균으로, vegetative cell일 때 막대모양을 가진다. C.diff는 설사와 대장염을 일으키는 세균으로 spore가 장에 진입하게 되면 독소를 분비할 수 있는 항생제에 감수성을 갖는 영양형(vegetative form)으로 전환되며 질병을 유발하게 된다. 즉 보호 수단으로 spore를 형성해 화학물질, 열과 같은 극한의 환경 조건으로부터 미생물을 보호하고 유리한 조건이 되면 영양세포로 분화한다.
2. Pasteurization

먼저 pasteurization 과정에서는 bacteria sample들을 RT와 60℃에서 각각 5분간 sterilization시켰다. 실험결과, RT에 둔 경우 colony가 많이 생성됐지만 60℃에서 열처리 한 경우 spore에서만 colony가 생성됐다. 문헌 조사 결과, C.difficile spore는 71℃까지 생존할 수 있고 제거하려면 85℃이상의 온도가 요구된다.
3. Filtration

filteration 과정에서는 syringe와 membrane filter를 결합하고 bacteria sample을 injection하여 filtering을 진행했다. 이때 0.2µm인 것과 1.2µm인 pore filter를 사용했고 각각 넣어 sterilization했다. 실험결과, filteration sample에서 pore size가 0.2µm, 1.2µm일 때 둘다 colony가 형성되지 않았다. 문헌조사 결과, vegetative cell size는 3~4µm이고 spore size는 평균적으로 0.5~0.7µm를 갖는다고 한다. 이로 인해 1.2µm에서 발견되지 않은 점이 이론상 결과와 다름을 알 수 있다.
4. Dilution

Viability test를 수행하기 위해 dilution을 진행했다. 기존의 균 sample은 10⁹CFU/ml인데 vegetative cell의 경우 PBS 900µl에 cell 100µl를 넣는 과정을 4번 반복해 10⁵CFU/ml가 되게 희석해주었다. Spore의 경우, 한번 희석하여 CFU/ml가 되게 해주었다. 희석한 이유는 균의 농도가 매우 높게 되면 일부는 colony를 형성하지 못하거나 colony끼리 겹쳐서 CFU 측정에 오차가 발생할 수 있기 때문이다.
5. Viability test

Dilution 이후 spreader를 이용해 vegetative cell sample은 BHI agar plate에, spore cell sample은 BHI+Taurocholic acid plate에 도포해주었다. BHI(Brain Heart Infusion) 배지는 미생물 성장을 위한 성장 배지로 영양이 풍부하다. 그리고 Taurocholic acid는 spore가 영양세포로 분화할 수 있게 도와준다.

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1. Spore sterilization for food processing

Spore sterilization is a critical process in food processing to ensure the safety and quality of food products. Spores, which are the dormant, resistant forms of certain bacteria, can survive many conventional sterilization methods and pose a significant risk of contamination if not properly eliminated. Effective spore sterilization techniques, such as high-temperature, high-pressure, or chemical treatments, are essential to eliminate these hardy microorganisms and prevent the growth of pathogenic or spoilage bacteria in food. The choice of sterilization method depends on the specific food product, its composition, and the target microorganisms. Proper validation and monitoring of the sterilization process are crucial to ensure consistent and reliable results. Implementing robust spore sterilization protocols is a fundamental step in maintaining the safety and integrity of processed foods.
2. Pasteurization

Pasteurization is a critical food processing technique that plays a vital role in ensuring the safety and extending the shelf life of a wide range of food and beverage products. By applying controlled heat treatment, pasteurization effectively eliminates pathogenic microorganisms, such as bacteria, viruses, and parasites, without significantly altering the nutritional and sensory properties of the food. This process is particularly important for perishable products like milk, juices, and certain prepared foods, where the presence of harmful microbes can pose a serious health risk. The specific time and temperature parameters of pasteurization are tailored to the specific food item, taking into account factors like the type of microorganisms present, the food's composition, and the desired shelf life. Proper implementation of pasteurization, along with appropriate storage and handling conditions, helps to maintain the quality and safety of food products, making it an essential tool in modern food processing and preservation.
3. Filtration

Filtration is a fundamental process in food processing that serves multiple important functions. It is used to remove unwanted particles, impurities, and microorganisms from food and beverage products, improving their clarity, purity, and safety. Depending on the specific application, different filtration techniques, such as membrane filtration, centrifugal separation, or adsorption, can be employed to achieve the desired level of purification. Effective filtration helps to extend the shelf life of products, maintain their sensory qualities, and ensure compliance with regulatory standards. In the case of beverages, filtration is crucial for removing yeast, bacteria, and other suspended solids, resulting in a clearer, more stable, and visually appealing final product. For dairy products, filtration is used to remove fat globules, casein, and other components, enabling the production of specialized dairy ingredients. Overall, filtration is a versatile and essential tool in modern food processing, contributing to the quality, safety, and consistency of a wide range of food and beverage products.
4. Dilution

Dilution is a fundamental technique in food processing that serves multiple important purposes. It is used to adjust the concentration of various components in food and beverage products, such as reducing the sugar or salt content, or increasing the water content to achieve the desired sensory and functional properties. Proper dilution is crucial for maintaining the safety and stability of food products, as it can help to control the growth of microorganisms, prevent the precipitation of certain compounds, and ensure the desired viscosity and texture. In the case of concentrated or highly viscous ingredients, dilution is necessary to facilitate their handling, mixing, and incorporation into final products. Additionally, dilution is often used in the preparation of food additives, flavorings, and other specialized ingredients, where precise control over the concentration is essential for consistent performance and quality. Overall, dilution is a versatile and indispensable tool in food processing, enabling the optimization of product formulations, the enhancement of safety and stability, and the achievement of desired sensory and functional characteristics.
5. Viability test

Viability testing is a critical component of food processing and quality control, as it helps to ensure the safety, quality, and stability of food products. These tests assess the ability of microorganisms, such as bacteria, yeast, and mold, to survive and grow under specific conditions, providing valuable information about the potential for spoilage or the presence of pathogenic microbes. Viability testing can be performed using a variety of methods, including plate counting, fluorescence-based assays, and molecular techniques, depending on the specific requirements of the food product and the target microorganisms. The results of these tests inform decisions about product formulation, processing conditions, storage, and shelf-life, allowing food manufacturers to implement appropriate control measures and ensure the delivery of safe, high-quality products to consumers. Robust viability testing protocols, combined with other quality control measures, are essential for maintaining the integrity and safety of the food supply, making it a crucial aspect of modern food processing and quality assurance.

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